The large molecular weight components of the clostridial glycine reductase system are separated by rechromatography on DEAE into two protein components, protein B and fraction C, which separately are enzymatically inactive. However, they combine to form the enzymatically active complex in the presence of protein A. Protein B was purified to a high degree, but fraction C still is heterogeneous. Protein B displays a high sensitivity to carbonyl reagents. Based on this observation, an independent assay for protein B that measures the release of tritium from 2-3H-glycine to water or the incorporation of tritium from 3H2O into glycine was developed. The formation of ammonia or ATP required the participation of all three protein components.